Halotag antibody

Determination of Nanoluc luciferase activity was measured using Nano-Glo Assay System following manufacturer's protocol (Promega, WI). In brief, 50 μl culture media was mixed with 50 μl of Nano-Glo Assay Reagent in a 96-well plate for an incubation period of 5 min. Then luciferase activity was measured using a Perkin Elmer Fusion-alpha-FP-HT universal microplate analyser. Detection of HaloTag was achieved either in live cells using HaloTag® TMR Ligand following manufacturer's protocol (Promega, WI) or in fixed cells using HaloTag antibodies (Promega, WI).

Both the Strep II/6X His tagged mNf1 cDNA and Halo tagged hNF1 cDNA were co-transfected into HEK293 NF1 null cells. Then, either the Strep tag affinity purification or Halo tag affinity purification was carried out on the co-transfected cells. Once neurofibromin was purified with either of the protocols, the remaining protein was utilized for Western blot and denatured at 95 °C for 5 min. Samples were loaded on Bio-Rad (Hercules, CA, USA) 4%–20% gradient gels (Cat. #: 4568094) and run at 100 V for 2 h. The gels were transferred onto a polyvinylidene difluoride (PVDF) membrane at 100 V for 2 h. Blots were probed overnight at 4 °C with either the neurofibromin antibody (Cell Signaling Technologies Cat. #: 14623S; Danvers, MA, USA), the His tag antibody (GenScript Cat. #: A00186; Nanjing, China), or the Halo tag antibody (Promega Cat. #: G921A; Madison, WI, USA). The next morning, the blots were washed, and then probed with the respective secondary (Rabbit = Neurofibromin; Murine = His tag and Halo tag). They were washed three more times before imaging using chemiluminescent substrate from BioRad (Cat. #: 170-5061; Hercules, CA, USA) per the manufacturer’s protocols.